A neurotherapeutic approach with Lacticaseibacillus rhamnosus E9 on gut microbiota and intestinal barrier in MPTP-induced mouse model of Parkinson’s disease

The gut microbiota plays a crucial role in neural development and progression of neural disorders like Parkinson’s disease (PD). Probiotics have been suggested to impact neurodegenerative diseases via gut-brain axis. This study aims to investigate the therapeutic potential of Lacticaseibacillus rhamnosus E9, a high exopolysaccharide producer, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of PD. C57BL/6 mice subjected to MPTP were fed L. rhamnosus E9 for fifteen days and sacrificed after the last administration. Motor functions were determined by open-field, catalepsy, and wire-hanging tests. The ileum and the brain tissues were collected for ELISA, qPCR, and immunohistochemistry analyses. The cecum content was obtained for microbiota analysis. E9 supplementation alleviated MPTP-induced motor dysfunctions accompanied by decreased levels of striatal TH and dopamine. E9 also reduced the level of ROS in the striatum and decreased the DAT expression while increasing the DR1. Furthermore, E9 improved intestinal integrity by enhancing ZO-1 and Occludin levels and reversed the dysbiosis of the gut microbiota induced by MPTP. In conclusion, E9 supplementation improved the MPTP-induced motor deficits and neural damage as well as intestinal barrier by modulating the gut microbiota in PD mice. These findings suggest that E9 supplementation holds therapeutic potential in managing PD through the gut-brain axis.


Bacterial strains
A previously described Lacticaseibacillus rhamnosus strain isolated from healthy infant feces (L.rhamnosus E9) was used in this study 43,53 .L. rhamnosus E9 was maintained at − 30 °C in MRS broth (Merck, Germany) with 10% (v/v) glycerol.Working cultures were prepared from frozen stocks by two sequential transfers in MRS broth and incubations were conducted statically at 37 °C for 24 h and 18 h, respectively.The culture at early stationary phase was harvested by centrifugation at 5000 rpm for 10 min at room temperature.The pellet was re-suspended in 0.85% NaCl (w/v) and the optical density at 600 nm (OD600) was determined to obtain a final concentration of 10 9 CFU/ml 32 .The final culture solution that was kept at 4 °C until the administration was enumerated daily on MRS agar to confirm the dose administered to the mice which is10 8 CFU/day.

Animals and experimental procedure
All the procedures involving mice were reviewed and approved by the Local Ethics Committee for Animal Experiments with the protocol #311 (Kobay D.H.L. A.S., Ankara, Turkey) and all experiments were performed in accordance with relevant guidelines and regulations.Additionally the study conducted adheres to the ARRIVE guidelines 54 .A total of sixty healthy male C57BL/6 mice (8-10 week-old) were obtained from and housed as one in each cage at Kobay D.H.L. A.S. facility by following the guide for the care and the use of laboratory animals.Housing conditions were controlled at 22 ± 2 °C, 65-70% relative humidity with a 12 h light/dark cycle.Mice were fed the same water and mouse chow ad-libitum throughout the study.A week after acclimation to their surroundings in testing room, the animals were randomly divided into 4 groups; each group (n:15) was administered daily 100 μL of either 0.85% NaCl (Control and MPTP) or 10 8 CFU/ml of L. rhamnosus E9 (E9) by oral gavage for fifteen days (Fig. 1).All mice except the Control groups were administered an intraperitoneal injection of 30 mg/kg MPTP-HCl (MedChemExpr, USA) daily for 5 consecutive days 55 .The Control groups were received equal volume of saline with the same procedure.All mice were sacrificed at the end of the experimental period, seven days after the first injection.After sacrifice, the cecum content was immediately placed on ice, and then kept at − 20 °C until processed for microbial DNA isolation.Animals in each group were randomly divided into three and the striatum of each animal was dissected out and rapidly snap-frozen for ELISA, preserved in 10% formaldehyde (Sigma-Aldrich, USA) for immunohistochemistry, or preserved in RNAlater (Ambion, USA) at 4 °C overnight and then at − 80 °C for RNA extraction.

Behavioral procedures
After preadaptation and training, behavioral tests for motor functions were performed until the sacrifice.Tests were applied at the same time in the same order.

Open-field
For general locomotor activity and exploratory behavior of the mice, open field test was performed 23,56 .Each mouse was placed in the middle of a squared plexiglass box (40 × 40 × 40 cm) and allowed to freely move for 10 min while being monitored by an overhead camera.The records were analyzed to calculate the total distance traveled (mm) during the observation period and the average speed (mm/s) using an animal tracking software (ToxTrac) 57 .

Catalepsy bar test
The catalepsy of each mouse was evaluated 58 .The mice were positioned individually with forelimbs on a 4 cmhigh horizontal bar and the maintenance time of the posture with fore/hindlimbs was recorded in seconds.The cut-off time was 180 s.

Wire hanging test
A wire-gripping test was performed to assess the whole body force 59 .Mice were suspended from a horizontal wire (30 cm high) by their forelimbs.Each mouse was scored from 1 to 4 based on the number of fore/hindlimbs used for gripping.Falling from the wire was scored as 0. The maximum time allowed was 60 s.

Immunohistochemistry
After sacrification, brain and intestinal tissues were dissected intact and fixed in 10% neutral buffered formaldehyde.Sections were taken from the brain tissues in the coronal plane and from the intestinal tissues in the luminal plane.After tissue processing, paraffin embedding was performed and 4 micron sections were obtained for immunohistochemistry. Histopathological sections were kept in an oven at 70 °C for 30 min.and deparaffinized.The sections were then incubated in a graded alcohol series (100%, 100%, and 95%) for 5 min each and then rinsed under running tap water for 5 min.Antigen retrieval was performed in pH 6 citrate solution or pH 9 tris/EDTA solution depending on the antigen.The Leica Bond Max Autostainer (Shandon, Frankfurt, Germany) was used with the appropriate Leica brand secondary antibody kit.Primary antibodies against tyrosine hydroxylase (TH) (1/100, Biolegend, San Diego, CA, USA), SOX-10 (1/200, Biolegend, San Diego, CA, USA), GFAP (prediluted, Boster Lab, Pleasanton, CA, USA), Zonula Occluden-1 (ZO-1) (1/200, Boster Lab, Pleasanton, CA, USA), and Occludin (1/100, Booster Lab, Pleasanton, CA, USA) were stained using Leica Bond Max Autostainer (Frankfurt, Germany).The evaluation of immunohistochemistry was performed using different methods according to the tissue characteristics and the staining pattern.The detail of the evaluation for each antibody as follows; TH, from each brain tissue, 100 × microphotographs were obtained (Olympus, cellSens software, Tokyo, Japan) from the demonstrative striatum region and pixels in these photographs were converted into numerical values in the Image J software ("color threshold" tool) developed by the NIH (US Department of Health).SOX-10, the luminal sections were examined under a light microscope and positive nuclear reactions in submucosal and myenteric plexus were manually counted.GFAP, expression in the submucosal and the myenteric plexus localization representing the luminal neuronal axis was examined.While microphotographs were taken, mucosa was removed from the sections to rule out cross-reactions and to obtain a more definite result (to reduce the error of digital analysis).For the staining intensities on the wall outside the mucosa, pixel calculation was performed using the image J software.Occludin and ZO-1, evaluation of the epithelial tight junction proteins was performed on the mucosa.The staining intensity in the photographs were scored from 1 to 4 (score 1: barely visible staining, score 2: faint staining, score 3: neither strong nor faint staining, score 4: strong positive reaction).

RNA isolation and gene expression analysis
Weighed brain and distal small intestine tissues were homogenized in ultra-pure-guanidine isothiocyanate (Sigma-Aldrich) using innuSPEED Lysis Tubes P in SpeedMill Plus (Analytik Jena, Germany).Total RNA was isolated and DNA contamination was removed using PureLink™ DNase Set (Invitrogen).Total RNA was converted into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to supplier's protocol.qPCR was performed using the primers shown at Table S1.AMPIGENE qPCR Green Mix Hi-ROX (Enzo Life Sciences) was used under the following conditions: 95 °C for 2 min for initial denaturation, followed by 5 s (40 cycles) at 95 °C and 30 s at 57 °C.Data were generated in the final step at 95 °C for 15 s and melting curves (65 to 95 °C) were acquired at the end for each primer set.Relative gene expression was calculated by 2 −ΔΔCt method by normalizing gene expression to β-actin 60 .

Enzyme-linked immunosorbent assay
Brain tissues for detection of dopamine and ROS level were homogenized in T-PER™ Tissue Protein Extraction Reagent and Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Scientific) using innuSPEED Lysis Tubes E in SpeedMill Plus (Analytik Jena, Germany) [61][62][63] .After centrifugation at max. speed for 5 min, the supernatant was used to carry out the experimental procedure as recommended by the manufacturer and the optical densities were measured at 450 nm.

DNA extraction and gut microbiota analysis
After weighing, the cecum content was homogenized in PBS and total DNA was extracted using the QIAamp Fast DNA Stool-Mini Kit (Qiagen Sciences, MD) as described previously 32 .Briefly, a mechanical cell-disruption step was included by 0.1 mm glass-beads (Sigma-Aldrich) and specimens were beaten at maximum speed for six 1-min using the SpeedMill Plus (Analytik Jena, Germany) with intervals of 2-min on ice.Subsequently, a heat treatment step was carried out at 95 °C for 5 min and DNA was extracted.After DNA was quantified by Nan-oDrop spectrophotometer (Epoch, BioTek), 16S rRNA sequencing was performed using the Illumina NovaSeq 6000 at BMlabosis (Ankara, Turkey).
The V3-V4 region was amplified for library preparation, using specific primers (F-5ʹTCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACGGGNGGC WGC AG-3ʹ, R-5ʹGTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTACHVGGG TAT CTA ATC C-3ʹ).The Nextera XT index kit (Illumina, San Diego, CA) was used to attach unique identifiers to both 5′ and 3′ ends.The amplicons with equal quantities were pooled after PCR products were cleaned up using AMPure XP magnetic beads.Paired end reads sequenced on NovaSeq 6000 system underwent a quality-filtering and were trimmed with quality cut-off of Phred score of 20 by DADA2.OTUs were generated using DADA2 and data analysis was assessed in QIIME2 framework.The sequences were submitted to the Sequence Read Archive (SRA) database with the accession number of PRJNA1015441.

Statistical analysis
The sample size was estimated to be five/group with 80% power (unpaired t-test; α = 0.05) to detect differences between the control and the treatment groups 21,64 .For microbiome, the statistical difference between groups was analyzed using the Mont Carlo test, a non-parametric test based on random permutations, in package ade4 generated by R 3.6.1 as described by de Carcer et al. [65][66][67][68] .The statistical difference of the between groups was evaluated with the randtest.betweenfunction in package ade4 of R 3.6.1.The zero (0) values were replaced with the limit of detection, which is assessed by the ratio of one to the lowest number of read in the data set.The Benjamini Hochberg procedure was applied to manage the false-discovery rate.The dominant genera that raised or diminished in abundance were identified by correspondence analysis in package ade4 of R 3.6.1 68 .Statistical difference for the rest of the analyses was performed with the Student's t test or the Wilcoxon rank sum test using JMP Pro (SAS Institute Inc., Cary, NC) and introduced as mean ± SD.Statistical difference was ascertained at a P value of ≤ 0.05.

Lacticaseibacillus rhamnosus E9 administration improved motor dysfunction in MPTP-induced mouse model of PD
Motor dysfunction is one of the important characteristics of PD, which reflects dopamine depletion 7,8 .Behavioral tests were applied to measure the motor dysfunction that occurs due to dopaminergic damage in the mouse brain after MPTP injection.The general locomotor activity and exploratory behavior of mice were tested by open field test.A standard bar test and a wire grip test were administered to evaluate catalepsy and whole body strength, respectively.The effect of MPTP was reflected in the behavioral tests one day after MPTP injection and the normal locomotor activity, the exploratory behavior, and the posture maintenance ability of the mice were impaired.These behavioral findings are consistent with the motor symptoms in MPTP-induced PD mouse models as part of their response to MPTP administration [69][70][71] .MPTP injection significantly decreased the locomotor activity and animals' exploratory behavior in both the MPTP and the probiotic groups.It can be seen in the open-field test with reduced average speed and overall traveled distance in 10 min (Fig. 2).However, E9 administration helped to recover the locomotor activities faster than the MPTP mice that were not received probiotics.Catalepsy and wire hanging test results (Fig. 2) showed that the total time to correct an imposed posture resulting from muscular rigidity and muscle strength were not affected in the E9 group as much as in the MPTP group (p < 0.05).These results show that E9 administration effectively alleviates the impaired motor functions in MPTP-induced mouse model of PD.In a study applying another strain of the same species as we used in this study (L.rhamnosus HA-114), probiotic treatment had no significant effect on behavioral deficits in an OHDA-induced PD mouse model, showing us the strain specific impact of probiotics 72 .On the other hand, there are studies stated that brain damage may not be reflected in behavioral tests to the same extent or that this reflection may vary depending on the behavioral test applied 55,[73][74][75] .

Lacticaseibacillus rhamnosus E9 administration attenuated dopaminergic neuronal death in MPTP-induced mouse model of PD
Dopaminergic neural damage occurring in PD patients leads to a decrease in dopamine levels, and therefore, in the levels of dopamine receptors, dopamine transporters, and the tyrosine hydroxylase enzyme (TH) that synthesizes dopamine 76 .Decrease in dopamine level is one of the pathological changes occurring in PD and TH, the rate-limiting enzyme of dopamine synthesis, plays a critical role in this pathology 7,8 .TH, hence, has been recognized as a biomarker of dopaminergic neurons and measured to validate the success of the PD animal models generated to examine PD pathology and to develop possible therapeutics 77,78 .After striatum was dissected from the brain tissues, levels of TH and dopamine were measured to evaluate the dopaminergic neuronal death in mice.(Fig. 3).A significant decrease in TH expression was detected in striatum of the MPTP mice, indicating an impaired dopamine synthesis by MPTP injection.Immunohistochemistry showed that TH expression that was decreased significantly (p < 0.05) after MPTP application (54 ± 11%) was increased by E9 administration (72 ± 16%).Similar results were seen in striatal TH gene expression as well.Additionally, dopamine level in the striatal tissues was decreased significantly (p < 0.05) after MPTP application in parallel with the level of TH enzyme, a dopamine producer.Dopamine in the mice administered E9 was not significantly different than the  76 .MPTP injection has been shown to reduce DAT levels in brain tissue of PD animals as part of the pathophysiology of PD 80 .In this study, the expression level of DR1, DR2, and DAT genes of the striatal tissues were examined.While the expression of DR1 significantly (p < 0.05) increased in the MPTP-induced mice, DR2 showed no statistical differences from the control in all samples evaluated (data not shown).Reports on modification in the level of dopamine receptors has been controversial in both PD animal models and PD patients 76,79,[81][82][83][84] .A decrease, an increase, or no change have been documented in MPTP-induced animal models of PD.The DAT expression in MPTP-induced mice was reported for the first time in this study and found to be significantly (p < 0.05) decreased in parallel with the loss of dopaminergic neurons in MPTP mice (Fig. 4).The expression levels of DR1 and DAT in mice administered E9 showed no significant differences compared to the control.

Lacticaseibacillus rhamnosus E9 administration reduced reactive oxygen species (ROS) formed in striatal tissue
Factors affecting the neural degeneration in sporadic PD patients include an increase in the production of free radicals and oxidative stress 7,8 .Reactive oxygen and nitrogen are mainly produced in mitochondria as a result of aerobic metabolism.Incomplete induction of oxygen and nitrogen causes the formation of radicals such as superoxide radical (O2-), hydrogen peroxide (H2O2), and nitric oxide (NO).These radicals can cause oxidative damage to proteins, membranes, and DNA 85 .Oxidative stress has found to be high in the brain tissues of PD patients 86 .Reactive oxygen species (ROS) in the brain was determined to evaluate oxidative damage resulting from inflammation.In the MPTP group, a significant (p < 0.05) increase was observed in the ROS level compared to the control group.It was not significantly different in mice administered E9 from the control.E9 reduced the level of ROS formed in the striatal tissue by MPTP injection (Fig. 4).ROS has been shown to correlate positively with the inflammation in the brain of PD mice 87 .Additionally, studies suggest that production of ROS leads to misfolding of α-syn in the brain and contributes to development of synucleinopathies including PD 88 .Lactobacilli administration has been previously shown to prevent α-syn aggregation in substantia nigra of MPTP-induced mice by inhibiting the oxidative damage and the inflammation in brain 87 .Further investigation of the impact of E9 on inflammation in MPTP-induced mouse model of PD would provide more insight into the mechanism of action by E9 on oxidative stress and neuroprotection.

Lacticaseibacillus rhamnosus E9 administration enhanced intestinal barrier integrity in MPTP-induced mouse model of PD
One of the gastrointestinal symptom commonly seen in PD patients is leaky gut syndrome due to increased intestinal permeability 2,89 .Pro-inflammatory cytokines and glial markers have been reported to increase in colonic biopsies collected from PD patients and that these levels correlate with the duration of the disease 90 .In addition to the intestinal inflammation, a significant increase in the intestinal permeability has been observed in PD patients compared to their controls 18 .A weakened intestinal barrier causes translocation of bacteria and bacterial components which leads to inflammation and oxidative stress in the enteric nervous system 91 .To assess changes in the impact on tight junction proteins (TJPs), that provide intercellular connection and strengthen the intestinal epithelium of the E9 probiotic strain, we examined the ileal expression of ZO-1 and Occludin proteins in MPTP mice after probiotic administration.We observed a significant (p < 0.05) decrease in expression of both ZO-1 and Occludin protein and gene in MPTP mice (Fig. 5).Occludin gene and protein expression in probiotic MPTP mice was detected at the same level as the control.E9 administration improved the intestinal integrity.The expression of TJPs was not different than the control.Enteric glial markers, GFAP and SOX-10, were also evaluated.Expression of SOX-10 was significantly (p < 0.05) elevated in MPTP mice while it was not different than control in E9 administered mice (Fig. 5).Although GFAP showed no statistical differences from the control in all samples evaluated (data not shown), SOX-10/GFAP ratio, which induces intestinal motor dysfunctions and contributes to enteric inflammatory responses, was increased from 0.9 to 2.8 in MPTP mice but it was 1.5 in E9 administered mice 92 .An upregulation in SOX-10 expression has been shown also in colon biopsies collected from PD patients 90 .While the increase in GFAP and SOX-10 levels have found to be in parallel with the level of inflammation, not enough data has been reported about the GFAP level in PD patients 92 .In another study, rotenone induced mice were received L. plantarum CCFM405 for 9 weeks and the gene expression of TJPs in colon was examined 70 .L. plantarum administration ameliorated the decrease in gene expression of ZO-1 and Occludin after rotenone application.It was emphasized that probiotics impact the brain-gut axis by rearranging the gut microbiota and that probiotic administration can be an alternative strategy in PD management.The decrease in the level of ZO-1 in the probiotic only group of mice could be related to the increased intestinal SOX-10 level which is associated with inflammation 92 .Pattern recognition receptors such as Toll-like receptors www.nature.com/scientificreports/(TLRs) contribute to the interaction between intestinal cells, gut microbiota, and the immune system 93 .They are involved in maintaining the tight junctions between intestinal epithelial cells 94 .The expression of TLRs in the intestine may differ depending on the probiotic strain due to both their different surface structure and their interaction with the commensals [95][96][97][98][99] .Accordingly, intestinal inflammation could be indirectly affected [100][101][102] .

Lacticaseibacillus rhamnosus E9 administration remodulated the cecal microbiota altered in MPTP-induced mouse model of PD
Chronic inflammatory state, an imbalance between pro-inflammatories and anti-inflammatories has been thought to be the main cause of PD, which accelerates aging and damages neural cells 85,103 .Gastrointestinal microbiota is one of the main factors affecting and even regulating the immune system in the host 104,105 .Gut microbiota is also known to communicate with the brain and this communication maintains the gastrointestinal and the nervous system homeostasis along with the immune system [13][14][15] .A disruption in the communication within the gut-brain axis is associated with various neurodegenerative diseases including PD 16 .As a matter of fact, clinical studies have revealed dysbiosis in the intestinal microbiota of PD patients [19][20][21] .Recent research shows that histological and behavioral findings in PD are associated with dysbiosis in the gut microbiome and the potential of probiotics to prevent PD progression is one of the latest innovations in the field of neurodegenerative diseases 70,106,107 .However, studies unrevealing the mechanism of probiotic action on PD pathogenesis with the association of the brain-gut axis is limited.In our study, we demonstrated the changes in the microbial composition in MPTP-induced mouse model of PD and impact of E9 administration on the altered cecal microbiota by MPTP injection using 16S rRNA sequencing.As a result of sequencing, a total of 1,776,677 filtered reads were obtained from the cecal content samples of 20 mice.The number of reads per sample ranged from 47,182-168,617 with an average number of 88,833.85reads/sample.After assigning the each read to a taxonomic level, 18 phyla, 26 classes, 54 orders, 108 families and 257 genera were identified.To assess whether sufficient sequence reads had been obtained to accurately determine the diversity of organisms present, Chao1 and Shannon index were computed (Fig. S1).Additionally, a principal coordinate analysis (PCoA) plots using weighted Unifrac distances for beta diversity were generated.
The cecum microbiota of the MPTP mice fed with/without E9 was evaluated at the 3.5 h after the last administration, the time lactobacilli bolus reaches the cecum, and compared with the healthy control 32 .A Monte-Carlo test with 10,000 replicates was utilized to identify the groups that were significantly (p < 0.05) different from each other.The overall microbial composition at the phylum level differed significantly (p < 0.05) from the control in the MPTP injected mice (Fig. 6).The dominant phyla in rank order of the cecum microbiota were Firmicutes and Bacteroidota, which constitute 84-90% of the total microbiome (Fig. 6 and Table S2).While the dominant phylum in healthy and E9 mice was Firmicutes with 48% and 45%, respectively, the abundance of Firmicutes decreased significantly (p < 0.05) to 41% in the MPTP mice.The dominant phylum was Bacteroidota with 48% in the MPTP mice.The predominance of Bacteroidota in control and E9 mice was determined as 37.9% and 38.5%, respectively and was not significantly different from each other.Firmicutes and Bacteroidota are the two major phyla detected in human and rodent gastrointestinal microbiome, and the ratio of these two phyla has been found to be relevant to human health 108 .The Firmicutes/Bacteroidota ratio has been shown to be reduced in patients with Crohn's disease, ulcerative colitis, and infectious colitis, where the intestinal epithelial barrier is damaged and general inflammation is stimulated 109 .In our study, while the Firmicutes/Bacteroidota ratio was 1.3 and 1.2 in control and E9 mice, respectively, it decreased to 0.9 in the MPTP mice, which was statistically (p < 0.05) different from the healthy control and E9 mice.E9 administration increased the Firmicutes/Bacteroidota ratio.In a study conducted with fecal samples obtained from PD patients, the microbial composition was compared with the healthy controls and a decrease in the abundance of Firmicutes but an increase in the Bacteroidata abundance was reported 110 .
The overall microbial composition at the genus level in MPTP injected groups differed significantly (p < 0.05) from each other (Figs. 7, 8, and Fig. S2).The dominant genera of the cecum microbiota at the genus level were Muribaculaceae, previously known as S24-7, and Lachnospiraceae_NK4A136_group, and these genera together constitute 31-46% of the total microbiota (Table S3) 111 .While the abundance of Muribaculaceae increased from 24.6 to 29.8% in MPTP mice, it decreased to 21.4% in mice fed with E9.Muribaculaceae, for which there is not much information in the literature, is known as mucus-derived monosaccharide users in the gut 111 .Similarly, microbiome analysis of fecal samples from MPTP-induced PD mice demonstrated the Muribaculaceae population to be increased 112 .The predominance of Lachnospiraceae_NK4A136_group was between 16.0% and 16.9% in control and MPTP mice; however, it decreased to 10% in the mice administered E9.In a study investigating the relationship between the motor dysfunctions in PD and the gut microbiota, Lachnospiraceae_NK4A136 abundance was found to be increased in patients experiencing motor complications during chronic dopamine replacement therapy 113 .The predominance of Colidextribacter, which decreased in E9-administered mice, was reported to be associated with aging and showed a significant increase in the murine microbiota during aging 114 .Additionally, they were positively correlated with cognitive fragility.The abundance of Clostridia_UCG-014, which was negatively correlated with the cognitive fragility in studies exploring the gut-brain axis, was decreased in MPTP mice in our study 114 .E9 administration restored back to the same level as the control.The level of Oscillibacter, which is also reported to have a negative correlation with cognitive performance and inflammation, decreased in MPTP mice, while it was restored back to the same level as the control in mice received E9 115 .Likewise, the predominance of Lactobacillus and Ligilactobacillus, previously belonged to Lactobacillus, were decreased in MPTP mice but increased in mice fed with E9 to the same level as the control 116 .These genera consist of strains commonly used as probiotics and various health benefits have been associated with their use as probiotics 95,[117][118][119] .Desulfovibrio, negatively correlated with irritable bowel syndrome, was found to be associated with PD 120 .In our study, it positively correlated with the level of TH enzyme responsible for dopamine synthesis.The increase in the abundance of Alloprevotella, reported to be positively correlated with dyskinesia, was decreased by E9 administration 121 .Butyricicoccus is a producer of butyrate, a short-chain fatty acid used by enterocytes as an energy source and suppressing inflammation as well as enhancing the intestinal barrier 122,123 .PD-derived fecal samples were found to contain less butyrate producing bacteria 20,110 .A study with clinical phenotyping approach including metabolomics and metagenomics in PD patients reported that low level of butyrate was associated with the worse postural instability gait-disorder scores in PD 123 .The Butyricicoccus abundance decreased in MPTP mice while increased in mice fed with E9.The predominance of Prevotellaceae Ga6a1 group, associated with anxiety like behavior in rats, was increased by MPTP injection but was decreased in E9 mice 124 .Based on Spearman's ρ correlation analysis TH level in striatum was positively correlated with Desulfovibrio and Clostridia_UCG-014 abundance, but negatively correlated with Allobaculum (p < 0.0001).Additionally, ileal SOX-10 level showed a negative correlation with Oscillibacter and Helicobacter abundance (p < 0.0001).These results suggest that the composition of the cecal microbiota, which was altered by MPTP injection and associated with dopamine synthesis, was likely modified and restored by E9 administration.
E9 strain used in this study is known to be a high EPS producer [43][44][45] .EPSs are shown to have immunomodulatory effect and regulate the gut microbiota and the dopaminergic system in mice [46][47][48][49][50] .Modulating the gut microbiota, taking role in the neurodegenerative diseases, by E9 could be associated with the ability of E9 to produce high amount of EPS known to have biological activities such as anti-proliferative and anti-inflammatory activity, and neuroprotective effect [43][44][45]51 Since the impact of EPSs on PD has not been studied yet, further investigation on E9 EPSs for their impact on immunomodulation and PD development could be significant to understand the mechanism of probiotic action.

Conclusion
In conclusion, we provide a comprehensive study investigating the effect of probiotics on PD with the association of the gut-brain axis.Overall our results indicated that L. rhamnosus E9 administration effectively exerted a neuroprotective effect on dopaminergic neural loss in MPTP-induced mouse model of PD.E9 improved the behavioral dysfunction of PD mice, inhibited MPTP-induced dopaminergic neural damage, oxidative stress, and gut barrier impairment and the neuroprotective mechanism of action involved the rearranging the gut microbiota.E9, an EPS producer lactobacilli strain, is a promising oral supplement as an alternative strategy for

Figure 1 .
Figure 1.Schematic illustration of the experimental timeline.

Figure 3 .
Figure 3. Impact of Lacticaseibacillus rhamnosus E9 administration on striatal Tyrosine hydroxylase (TH) protein (A, B) and gene (C) expression level and dopamine level (D) in MPTP-induced mouse model of PD compared to the control.MPTP mice were received (i.p.) 30 mg/kg MPTP-HCl daily for 5 consecutive days (MPTP and MPTP + P).Probiotic mice were administered 1 dose (10 8 CFU/mouse/day) daily of L. rhamnosus E9 for fifteen days and sacrificed after the last dose (P and MPTP + P). *p ≤ 0.05, control vs MPTP (n:4-5/group).

Figure 6 .
Figure 6.Comparison of predominant phyla in the microbiota of mice cecums.(A) Change of individual mouse cecum microbiota at the phylum level after MPTP and/or probiotic application.(B) Predominant phylum in the cecum microbiome of mice and pair wise comparison of each group.Statistical p-values were assessed using a Monte-Carlo test with 10,000 replicates.MPTP mice were received (i.p.) 30 mg/kg MPTP-HCl daily for 5 consecutive days (MPTP and MPTP + P).Probiotic mice were administered 1 dose (10 8 CFU/mouse/ day) daily of L. rhamnosus E9 for fifteen days and sacrificed after the last dose (P and MPTP + P). *p ≤ 0.05, (n:5/ group).

Figure 7 .
Figure 7.Comparison of predominant genera in the microbiota of mice cecums.(A) Change of individual mouse cecum microbiota at the genus level after MPTP and/or probiotic application.(B) Predominant genera in the cecum microbiome of mice and pair wise comparison of each group.Statistical p-values were assessed using a Monte-Carlo test with 10,000 replicates.MPTP mice were received (i.p.) 30 mg/kg MPTP-HCl daily for 5 consecutive days (MPTP and MPTP + P).Probiotic mice were administered 1 dose (10 8 CFU/mouse/day) daily of L. rhamnosus E9 for fifteen days and sacrificed after the last dose (P and MPTP + P). *p ≤ 0.05, (n:5/group).